Antiphospholipid syndrome (hereinafter, referred to as “APS”) is a general term for a group of diseases which have antiphospholipid antibodies in the blood and present clinical symptoms such as arteriovenous thrombosis and habitual abortion (see Mika Yoshida et al., The Japanese Society for Laboratory Hematology, 2008, 9 (1):69-76). Antiphospholipid antibodies (hereinafter, referred to as “aPL”) is a general term for autoantibodies which are bound to phospholipids or a complex of phospholipids and proteins (see Tatsuya Atsumi et al., The Japanese Society on Thrombosis and Hemostasis, 2008, 19 (3):329-332).
There are various antibodies aPL and the name of antibodies is given according to phospholipids recognized by the antibodies or phospholipid-binding proteins. Examples of the antibodies aPL include anti-cardiolipin antibodies (aCL), anti-β2 glycoprotein I antibodies (aβ2GPI), phosphatidylserine-dependent anti-prothrombin antibodies (aPS/PT), and lupus anticoagulant (hereinafter, also referred to as “LA”). aCL, aβ2GPI, and aPS/PT are detected by enzyme immunoassay (ELISA) and LA is detected by prolongation of phospholipid-dependent clotting time (see Masahiro Ieko et al., The Japanese Society on Thrombosis and Hemostasis, 2007, 18 (3):226-233).
LA is defined as “an immunoglobulin that inhibits phospholipid-dependent blood clotting reactions without inhibiting individual clotting factor activities. It is also considered that LA is an autoantibody that inhibits phospholipids themselves in the phospholipid-dependent blood clotting reactions.
Currently, the inspection standard of LA is as follows:    1) Prolongation of clotting time is observed in screening inspection for measuring phospholipid-dependent clotting times such as activated partial thromboplastin time (APTT), dilute Russel viper venom time (dRVVT), and kaolin clotting time (KCT);    2) The prolongation of clotting time is not improved even if mixing assay with plasma from healthy donors is performed;    3) It is confirmed that the clotting time is shortened by adding excessive phospholipids (test for assessing phospholipid dependence).
Finally, a subject is diagnosed to be LA-positive by excluding obvious clotting abnormalities such as inhibitors of clotting factors and influences of anticoagulants such as heparin (see Mika Yoshida et al., Journal of the Japanese Society for Laboratory Hematology, 2008, 9 (1):69-76).
One of the tests for assessing phospholipid dependence of LA is a method of assessing the presence of LA in a specimen including steps of: measuring the clotting time using a clotting time-measuring reagent which contains a low concentration of phospholipid and a clotting time-measuring reagent which contains a high concentration of phospholipid; and confirming the prolongation of clotting time depending on the phospholipid concentration based on a ratio of the clotting times obtained from the reagents.
The present inventors have completed a reagent kit capable of detecting LA at high sensitivity by adjusting the composition of phospholipid contained in a reagent (see U.S. Patent Publication No. 2004/0091952, U.S. Patent Publication No. 2005/0175983, and Japanese Patent Application Laid-Open (JP-A) No. 2006-133002).
Although LA can be detected by the above methods, prolongation of clotting time may be observed even if the reagent which contains a high concentration of phospholipid is used. That is, in the case of using the conventional methods, even a ratio of clotting time in an LA-positive specimen may be equal to that of a negative specimen. Thus, it is difficult to clearly separate an LA-positive specimen group from an LA-negative specimen group.